Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms

Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 72–77

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Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms Ploypat Niyomploy a , Chantragan Srisomsap b , Daranee Chokchaichamnankit b , Nawaporn Vinayavekhin c , Aphichart Karnchanatat d , Polkit Sangvanich c,∗ a

Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand c Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand d Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand b

a r t i c l e

i n f o

Article history: Received 22 July 2013 Received in revised form 18 October 2013 Accepted 23 October 2013 Available online 20 November 2013 Keywords: Superoxide dismutase Two-dimensional gel electrophoresis Liquid chromatography–mass spectrometry (LC–MS) Stemona tuberosa

a b s t r a c t Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant metalloenzyme [1]. The different isozymes are currently classified according to the metal cofactor in their active sites into the four main and broad types of CuZn-SOD, Mn-SOD, Fe-SOD and Ni-SOD [2–5]. SODs have an important role in catalyzing the destruction of superoxide radicals, which are cytotoxic agents to cell membranes, DNA and other biomolecules, to hydrogen peroxide and oxygen [6–8]. In general, SODs have been found in microorganisms, animals and plants. The SODs used as antioxidant agents for applications in medicine, and in the cosmetic, chemical and food industries are currently sourced and extracted (or cloned and recombinant produced) from plants [9]. Furthermore, at least some SOD isoforms

∗ Corresponding author. Tel.: +66 22187637. E-mail address: [email protected] (P. Sangvanich). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.10.035

in plants are involved in the defense against pathogens and in signaling responses for various stresses. Since the different SOD isoforms in plants show different responses to infection and stress [10–12], the level of expression of specific SOD isozymes in key tissues can be used as an indicator to determine the growth stage or stress/infection circumstances of that plant [13]. Currently several methods have been reported for SOD detection with one of the popular methods being detection by zymograms. This method detects active enzymes across the spectrum of the four broad types of SODs, rather than, for example, immunological detection which often cannot discriminate between enzymically active and inactive forms, and can only detect those isoforms with conserved epitopes. The original zymogram detection method for SODs was created by Beauchamp and co-workers and used native polyacrylamide gel electrophoresis (Native-PAGE) followed by enzymatic staining for SOD activity [14]. There are two combined reactions in the SOD activity assay. The first is the autooxidation from riboflavin (oxidizing agent) and the second is the

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riboflavin/nitroblue tetrazolium (NBT) reduction, which uses NBT as a chromogenic substrate. The SOD enzyme can be determined as an achromatic zone on Native-PAGE because the enzyme inhibits NBT reduction [15], but Native-PAGE does not allow determination of valuable information, such as the subunit molecular weight (MW ), nor discriminate between isozymes with a different MW or isoelectric point (pI). However, the simultaneous detection of SOD activity and evaluation of their MW on one dimensional denaturing sodium dodecyl sulphate-PAGE (1D-SDS-PAGE) zymograms cannot separate the different SOD isoforms that share a similar MW but differ in their pI. Two-dimensional gel electrophoresis (2D-GE) is one of the important techniques for protein detection and is often coupled with tryptic digestion and mass spectrometry (MS) to identify the resolved protein(s) [16]. Proteins are separated by 2D-GE according to their pI in the first dimension and their apparent MW (three dimensional size) in the second dimension, using isoelectric focusing (IEF) and denaturing SDS-PAGE, respectively [17]. Consequently, proteins with a different pI but similar MW , as well as those that differ in apparent MW , can be resolved even in complex samples, such as different SOD isozymes from a whole organism or tissue extracts [18]. According to 2D-GE theory, the capacity of resolution for the method is up to 10,000 different proteins, and it can detect less than 2 ng per spot [17]. However, due to the denaturing running conditions of 2D-GE (SDS, dithiothreitol and urea plus sample heating) this method is typically unsuitable for SOD (and many other enzymes) zymograms due to the inactivation of the enzyme [16]. Accordingly, in this research, a 2D-GE approach was developed that appears to maintain SOD activity and so it is suitable for zymogram based detection of enzymically active SOD isoforms from tissue extracts. The method combined a non-denaturing IEF first dimension and SDS-PAGE second dimension, before staining duplicate gels with either Coomassie blue or SOD activity. Finally, in-gel trypsin digestion and peptide extraction was coupled with liquid chromatography tandem MS (LC–MSMS) of a known standard (CuZn-SOD from bovine erythrocytes) and an experimental tissue extract (crude total protein extract from the root of the medicinal plant Stemona tuberosa Lour.) to evaluate the reliability and usability of the method.

2. Methods and materials 2.1. Isolation and extraction of SOD from S. tuberosa Fresh tubers of S. tuberosa (∼3 kg, fresh weight) were purchased from Chatujak market, Bangkok, Thailand, in June 2009. A voucher specimen (BK 244965) is deposited at the Sirindhorn Bangkok Herbarium, Bangkok, Thailand. The fresh roots of S. tuberosa were peeled, cut into small cubes and homogenized in 5 L of extraction buffer (0.1 M NaCl, 20 mM phosphate buffer pH 7.2) at 4 ◦ C. The suspension-solution mixture was stirred at 4 ◦ C overnight before being clarified by centrifugation (10,178 × g, 30 min, 4 ◦ C) and harvesting the supernatant. Ammonium sulfate was then added to the supernatant to 90% saturation and left overnight at 4 ◦ C, before harvesting the precipitated material by centrifugation (15,904 × g, 30 min, 4 ◦ C), dissolving the precipitate in 400 mL de-ionized water, and dialyzing against 4–5 changes of 1000 mL each of 20 mM phosphate buffer pH 7.2 over 18 h at 4 ◦ C. The dialyzed extract was then freeze-dried to yield the dark brown crude protein extract, from which the IC50 value of the SOD preparation was determined as reported [19]. In addition, a commercial preparation of the CuZn-SOD isozyme from bovine erythrocytes (Merck, USA), a homodimer enzyme of 32.50 kDa MW and a pI of 4.95, was used as a positive control.

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2.2. Resolution and detection of SOD isozymes 2.2.1. One dimensional reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D-SDS-PAGE) Reducing 1D-SDS-PAGE was run according to the modified method as previously reported [19,20]. Either 12.5 ␮g (per track of the gel) of the reference pure CuZn-SOD homodimer enzyme from bovine erythrocytes, as a positive control, or 25 ␮g (per track of the gel) crude protein extract from S. tuberosa tubers was mixed with reducing sample buffer (62.5 mM Tris–HCl pH 6.8, 14.4 mM 2-mercaptoethanol, 10%, v/v glycerol, 2%, w/v SDS) and 1% (w/v) bromophenol blue at room temperature and then subjected to duplicate 1D-SDS-PAGE resolution using a 10% or 12.5% (w/v) acrylamide resolving gel for the bovine erythrocyte CuZn-SOD or the crude protein extract from S. tuberosa, respectively. After electrophoresis, one of each pair of duplicate gels was stained with Coomassie brilliant blue G-250 to visualize the protein bands, whilst the other, as a SOD zymogram, was washed and stained for SOD enzyme activity (see Section 2.3) to determine the presence of enzymically active SOD. Crude protein extracts were also mixed with reducing sample buffer or non-reducing sample buffer (62.5 mM Tris–HCl pH 6.8, 10%, v/v glycerol and 1%, w/v bromophenol blue) and applied (25 ␮g per track of the gel) to a 1D-SDS-PAGE and 1D-Native-PAGE (both 12.5%, w/w gel resolving gel), respectively, for comparison of the SOD activity level. 2.2.2. Non-denaturing two-dimensional polyacrylamide gel electrophoresis (2D-GE) The reference CuZn-SOD isozyme from bovine erythrocytes (40 ␮g per gel), and the crude protein extract from S. tuberosa (150 ␮g per gel) were dissolved in lysis solution (40 mM Tris, 4%, w/v 3-(3-cholamidopropyl) dimethylammonio-1propanesulfonate (CHAPS), 1 mM ethylenediaminetetraaceticacid (EDTA), 2%, v/v immobilized pH gradient (IPG) buffer, 10%, v/v glycerol and 1%, w/v bromophenol blue), vortexed every 30 min for 1–2 h and left on ice as previously reported [21]. Each protein sample was loaded onto duplicate 7-cm, pH 3–6 IPG gel strips (Bio-RAD Laboratories, CA, USA) and left overnight at room temperature (RT). The first-dimensional IEF electrophoresis was performed at 4 ◦ C on a Pharmacia LKB Multiphore II system at 300 V for 1200 Vh and then increasing the voltage step wise to 1000 V for 300 Vh, 5000 V for 4500 Vh and 5000 V for 1000 Vh. After IEF, the IPG strips were equilibrated in equilibration buffer (50 mM Tris–HCl buffer pH 6.8, 6 M urea, 1%, w/v SDS, 30%, v/v glycerol, 1%, w/v dithiothreitol (DTT)) and were alkylated with equilibration buffer containing 2.5% (w/v) iodoacetamide (IAA). Finally, the equilibration IPG strips were separated in the second dimension using the reducing SDS-PAGE and then the duplicate gels stained for protein and SOD activity, respectively, as described in Sections 2.2.1 and 2.3, respectively. 2.3. Gel washing procedures and SOD staining activity assay After electrophoresis, the 1D-SDS-PAGE or 2D-GE gel was washed as reported previously [22,23]. In brief, the gel was first soaked twice in 100 mL of 25% (v/v) isopropanol in 0.01 M Tris pH 7 for 10 min each at RT to remove the SDS. The gel was then washed twice with 100 mL of 2 ␮M ZnCl2 /0.01 M Tris pH 7 for 10 min each at RT to remove the isopropanol, then twice with 100 mL of 0.1 M Tris pH 7 for 20 min each, and once with 100 mL of 0.01 M Tris pH 7 for 10 min, all at RT. 2.4. SOD isozyme identification by in-gel trypsin digestion and tryptic peptide mass spectrometry analysis Protein spots on the Coomasie stained 2D-GE that matched the spots on the SOD activity zymogram in the paired duplicate gel

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were excised and washed three times in 100 ␮L distilled water before cutting into small pieces. The gel pieces were then washed with 0.1 M NH4 HCO3 in 50% (v/v) acetonitrile (ACN) at 30 ◦ C for 20 min to de-stain, dried in a Speed Vacuum and incubated in 50 ␮L of buffer solution (0.1 M NH4 HCO3 /10 mM DTT/1 mM EDTA) for 45 min at 60 ◦ C to reduce the gels. The supernatant was discarded and the gels incubated in 50 ␮L of 100 mM IAA in 0.1 M NH4 HCO3 for 30 min at RT in the dark. The supernatant was removed and the gels were washed three times with 50 ␮L each of 0.05 M Tris–HCl pH 8.5 in 50% (v/v) ACN and then dried in a Speed Vacuum. Tryptic digestion was performed by the addition of 30 ␮L of trypsin digestion buffer (0.05 M Tris–HCl pH 8.5, 0.1 ␮g/␮L trypsin in 1%, v/v acetic acid, 10%, v/v ACN and 1 mM CaCl2 ) to the excised region of the gel containing the desired spot and incubating at 37 ◦ C overnight. The reaction was stopped by adding 20 ␮L of 2% (v/v) trifluoroacetic acid and incubating for 30 min at 60 ◦ C, and then the supernatant was harvested. The gels were then extracted three times with 50 ␮L 2% (v/v) trifluoroacetic acid and 0.05 M Tris–HCl pH 8.5 containing 1 mM CaCl2 and 2% (v/v) formic acid, and for 10 min at 30 ◦ C on a shaker (1500 rpm), and then sonicated for 5 min. The harvested solutions were pooled and dried in a Speed Vacuum for further characterization by nano-LC–MSMS as detailed in Section 2.5. 2.5. SOD characterization by liquid chromatography tandem mass spectrometry (LC–MSMS) Each tryptic digested zymogen spot protein (putative SOD enzyme, see Section 2.4) was mixed with 0.1% (v/v) formic acid before being analyzed by nano liquid chromatography-electrospray ionization quadrupole-time of flight MS (nano-LC–ESI–MSMS) using an EASY-nLCII spectrometer coupled with a MicroTOF QII (Bruker, Germany). The tandem mass spectra of the tryptic peptides were searched from Mascot database (http://www.matrixscience.com/cgi/search formpl?FORMVER=2& SEARCH=MIS). The precursor and MSMS tolerances were set to ±1.2 Da and ±0.6 Da, respectively. 3. Results and discussion 3.1. Evaluation of the 2D-GE method using the bovine erythrocyte CuZn-SOD isozyme as a known standard The CuZn-SOD from bovine erythrocytes, a known homodimer with a pI of 4.95 and MW of 32.5 kDa, was used to determine the efficiency of the developed 2D-GE method for the detection of the SOD enzyme. Fig. 1 shows a representative 1D-SDS-PAGE and 2D-GE resolution gel zymogram of the CuZn-SOD isozyme from bovine erythrocytes after staining for SOD activity (Fig. 1A and B) or for protein (Fig. 1C and D). The 1D-SDS-PAGE resolution revealed a strong proteinband at ∼15–16 kDa (Fig. 1C) with a weak SOD activity (Fig. 1A), which presumably represents the monomer (15–16 kDa). However, the intensity of this protein spot is markedly reduced in the 2D-GE (Fig. 1D, spot 6), and showed no detectable SOD staining activity (Fig. 1B). This is potentially due to the renaturation of the monomer to homodimeric units. The 1D-SDS-PAGE gel also revealed three protein bands at around 55–58 and >66 kDa (Fig. 1C), at least two of which had a strong SOD activity in the zymogram (Fig. 1A). These were also potentially found in the 2D-GE as two spots at 55–58 kDa with a pI of 4.95 (Fig. 1D, spots 1 and 2) that also displayed SOD activity (Fig. 1B). These may be the homodimer but in native forms, where the abnormal (low) levels of bound SDS reduce the charge density and so accounts for the apparently higher MW in terms of the gel

Table 1 Comparison of the reported SOD activity (as IC50 values) from 10 plants. Plants

IC50 value (␮g/mL)

Reference

Wheat germ Alfalfa leaf Sphenostylis stenocarpa Pea (F2) Gnetum gnemon (Gg-AOPI) Melon Hedychium coronarium Kaempferia galanga Linn. Zingiber officinale Roscoe. Curcuma zedoaria Roscoe. Stemona tuberosa

250 700 >1000 1000 40 800 0.729 ± 0.008 0.681 ± 0.003 0.432 ± 0.003 0.259 ± 0.006 0.039 ± 0.006

[25] [26] [27] [28] [29] [30] [31] [31] [31] [31] Fig. S3

mobility [24]. If so, although these results show an advantage of the method in that it can maintain the native form of the SOD enzyme (or allow its partial renaturation) even though the second dimension was run under a denaturing condition, it also suggests that the discrimination of isoforms by MW , and the assignment of MW from the 2D-GE are both unreliable. Accordingly, to ascertain if the two large Coomassie blue stained spots on the 2D-GE gel (spots 1 and 2 in Fig. 1D) were the CuZn-SOD, the spots were cut out, digested by trypsin and the resultant peptide fragment sequences identified by nano-LC–EIS–MSMS were used to search the Mascot database to identify their likely source. Both spots 1 and 2 matched the Cu-Zn SOD from Bos taurus with an amino acid match of 59/59, which over the 152 amino acids gives a 38.8% sequence coverage (Fig. 2A and Supplementary Table S1). 3.2. SOD isoform separation from a crude protein extract of S. tuberosa tubers To evaluate the efficiency of this 2D-GE method in real situations, the crude protein extract from S. tuberosa tubers was used as a model system. The initial choice of S. tuberosa was based upon its SOD activity, since the derived SOD activity (Supplementary Figs. S1 and S2) had a higher (lower IC50 value in the NBT reduction assay) than that reported in nine other plants (Table 1). The crude protein extract (150 ␮g) from S. tuberosa was initially resolved by 2D-GE using a pH 3–10 linear IEF strip (Fig. 3). However, most of the proteins, including those that displayed SOD activity, were found to be in the acidic zone resulting in poor resolution. To solve this problem, a narrow range linear IEF strip of pH 3–6 was used in the first dimension IEF to improve the resolution of the individual SOD proteins from each other and from the other non-SOD active proteins. After comparing the spots on the gel stained with Coomassie blue with those tested for SOD activity, three spots that potentially represent different (active) SOD isoforms were revealed. Two of these had an apparently similar MW of 53 kDa but slightly different pI values at 4.25 and 4.95 (spots 3 and 4 in Fig. 4D, respectively), while the third was smaller at 38 kDa and more acidic with a pI of 4.0 (spot 5 in Fig. 4D). These three SOD-active spots (spots 3–5) were excised from the corresponding Coomassie blue stained 2D-GE, digested with trypsin and then analyzed via nanoLC–ESI–MSMS. However, only spots 3 and 5 could be identified, presumably because the limited data in the plant protein sequence databases prevented matches of the tryptic peptides from spot 4. Those from spot 3 matched to the CuZn-SOD from Ananas comosus at 25/25 amino acids (a coverage of 16.4% over the total 152 amino acids), whilst spot 5 matched to the CuZn-SOD from Solanum lycopersicum and Zantedeschia aethiopica at 10/10 amino acids (a coverage of 6.6% over the total 152 amino acids) (Fig. 2C and D, respectively). The results from the 2D-GE coupled with SOD staining activity, therefore, suggest the presence of at least three different active SODs in S. tuberosa. More importantly,

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Fig. 1. Representative (A and C) 1D-SDS-PAGE and (B and D) 2D-GE of the CuZn-SOD isozyme (12.5 and 40 ␮g, respectively) from bovine erythrocytes after staining for (A and B) SOD activity or (C and D) Coomasie blue for protein. Note that the spot appearing at ∼30 kDa with pI ∼ 4.0 in C and D corresponds to the carbonic anhydrase enzyme within the commercial SOD standard.

(A) 1 51 101 151

(B) 1 51 101 151

MATKAVCVLK GDGPVQGTIH FEAKGDTVVV TGSITGLTEG DHGFHVHQFG DNTQGCTSAG PHFNPLSKKH GGPKDEERHV GDLGNVTADK NGVAIVDIVD PLISLSGEYS IIGRTMVVHE KPDDLGRGGN EESTKTGNAG SRLACGVIGI AK

MVKAVAVLGS SEGVKGTIYF TQEGDGPTTV TGSISGLKPG LHGFHVHALG DTTNGCMSTG PHFNPAGNEH GAPEDETRHA GDLGNVTVGE DGTVNVNIVD SQIPLSGSNS IIGRAVVVHA DPDDLGKGGH ELSKTTGNAG GRVACGIIGL QG

(C) 1 51 101 151

MVKAVAVLNS SEGVSGTYLF TQVGVAPTTV NGNISGLKPG LHGFHVHALG DTTNGCMSTG PHYNPAGKEH GAPEDEVRHA GDLGNITVGE DGTASFTITD KQIPLTGPQS IIGRAVVVHA DPDDLGKGGH ELSKSTGNAG GRIACGIIGL QG

(D) 1 51 101 151

MVKAVAVLTG SEGVQGTVFF AQEGEGPTTI TGSLSGLKPG LHGFHVHALG DTTNGCMSTG PHFNPAGKEH GAPEDGNRHA GDLGNVTVGE DGTVNFTVTD SQIPLTGLNS VVGRAVVVHA DSDDLGKGGH ELSKTTGNAG GRLACGVIGL QA

Fig. 2. Deduced amino acid sequences of (A) the CuZn-SOD isoforms and (B–D) the matches to the tryptic peptide sequences of the SOD isozymes from a crude protein extract of S. tuberosa tubers. Matching amino acid sequences (bold) of the tryptic peptides for (A) B. taurus CuZn-SOD spots 1 and 2 (see Fig. 1D) with the sequence from the bovine (B. taurus) erythrocyte CuZn-SOD. For peptides from S. tuberosa, the putative SOD isoforms matched with the CuZn-SOD sequence from (B) Ananas comosus (Fig. 4D, spot 3), and from (C) Solanum lycopersicum and (D) Zantedeschia aethiopica (Fig. 4D, spot 5).

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Fig. 3. Active SOD isozymes resolved and detected in the crude protein extract from S. tuberosa. Crude protein extract (150 ␮g) after 2D-GE resolution with a broad range pH (3–10) IEF strip in the first dimension and stained for (A) SOD activity or (B) with Coomassie blue for protein.

Fig. 4. (A and B) 1D-SDS-PAGE and (C and D) 2D-GE resolution of the crude protein extract (40 and 150 ␮g, respectively) from S. tuberosa after staining for (A and C) SOD activity and (B and D) total proteins by Coomassie blue.

the LC–MSMS results reveal the capability of this method in separating two different SOD isozymes from each other (Fig. 4, spots 3 and 4 with a similar MW but different pI). 3.3. The key feature for improving the developed 2D-GE method resolution The 2D-GE method developed for the detection and discrimination of active SOD isozymes used a non-denaturing IEF first dimension (IEF-focusing) and an apparently non-fully denaturing SDS-PAGE second dimension. For the IEF-focusing step, Tris and EDTA were used in the lysis solution and rehydration buffer instead of urea, thiourea and DTT. Since the cell membrane structure is maintained by calcium and magnesium ions, their chelation by EDTA will destabilize the cell membrane, whilst the Tris buffer maintains a stable pH for SOD activity after cell lysis. In addition, the salts obtained from the SOD extraction process can disturb the electrophoresis process giving a poor protein focusing in the IEF. Accordingly, the crude protein extract from S. tuberosa was dialyzed against distilled water, but this was still found to be insufficient (data not shown). Increasing the IEF time (Vh) moves the

salt ions to the end of the strip and was found to improve the resultant resolution (Supplementary Fig. S2). Thus, the appropriate buffer and prolonged IEF electrophoresis time are important steps to improve the resolution of the non-denaturing IEF-focusing step. Another step to increase the zymogram resolution was the gel washing steps after the SDS-PAGE to remove the bound SDS before staining for SOD activity. The presence of SOD interferes with the SOD staining activity and produces an irregular purple background on the gel. The reason for this observation remains unknown, although it is possible that SDS might inhibit SOD from binding to and reacting with superoxide radicals, thereby allowing the reduction of NBT to a deep blue-colored Formazan dye, even when SOD is present [15]. The removal of SDS by isopropanol, as previously reported [22,23], was used in the first wash, followed by two ZnCl2 solution washes to remove the isopropanol and then soaking the gel sequentially with 0.1 M and 0.01 M Tris–HCl (pH 7). This may also lead to renaturation of (some of) the gel-bound SOD monomers to functional homodimers and so restore (some of the) SOD activity [24].

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4. Conclusion Here, the first developed 2D-GE method for SOD isozyme detection was reported. The method was successfully applied to separate SOD isozymes that differ in pI and/or MW from a complex protein mixture, maintaining at least some of the different isoforms of SOD in the native form of the enzyme and/or allowing renaturation of some non-permanently denatured monomers to form active homodimers, even though the second dimension was run under denaturing conditions. So far, there has been no report on using 2D-GE coupled with SOD staining activity. However, this developed method could prove to be useful to many SOD-related research areas, including the use of specific isozymes as markers of plant stress responses, the identification of SOD from novel sources and the evaluation of their pI for determining appropriate resins and buffer for the large scale extraction and enrichment in order to obtain purified SOD enzymes in a timely manner. Acknowledgements The authors thank the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0013/2552), the 90th Anniversary of Chulalongkorn University, and the National Research University Project of CHE; and the Ratchadaphiseksomphot Endowment Fund (Aging Society Cluster CU-56-AS05 and Advanced Material Cluster CU56-AM09) for financial support of this research. We also gratefully thank Dr. Apaporn Boonmee for her kind advice and guidance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2013.10.035. References [1] Y. Wang, K. Osatomi, Y. Nagatomo, A. Yoshida, K. Hara, Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthysolivaceus), Comp. Biochem. Physiol. B 158 (2011) 289–296. [2] M.P. Babitha, H.S. Prakash, H. ShekarShetty, Purification and partial characterization of manganese superoxide dismutase from downy mildew resistant pearl millet seedlings inoculated with Sclerospora graminicola, Plant Sci. 163 (2002) 917–924. [3] D. Vyas, S. Kumar, Purification and partial characterization of a low temperature responsive Mn-SOD from tea (Camellia sinensis (L) O. Kuntze), Biochem. Biophys. Res. Commun. 329 (2005) 831–838. [4] H.D. Youn, E.J. Kim, J.H. Roe, Y.C. Hah, S.O. Kang, A novel nickel-containing superoxide dismutase from Streptomyces spp., Biochem. J. 318 (1996) 889–896. [5] A.F. Miller, Superoxide dismutase: active sites that save, but a protein that kills, Curr. Opin. Chem. Biol. 8 (2004) 162–168. [6] C.L. Fattman, J.J. Enghild, J.D. Crapo, L.M. Schaefer, Z. Valnickova, T.D. Oury, Purification and characterization of extracellular superoxide dismutase in mouse lung, Biochem. Biophys. Res. Commun. 275 (2000) 542–548.

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